Vol. 282, Issue 1, R156-R165, January 2002
Expression and control of C-type natriuretic peptide in rat
vascular smooth muscle cells
Geoffrey E.
Woodard,
Juan A.
Rosado, and
John
Brown
Physiological Laboratory, University of Cambridge, Cambridge CB2
3EG, United Kingdom
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ABSTRACT |
C-type natriuretic peptide (CNP) is a
member of the natriuretic peptide family mainly distributed in the
central nervous system. CNP is also produced and secreted by the
endothelium and inhibits vascular smooth muscle cell proliferation. We
have reported that endothelial damage stimulates only transiently
vascular smooth muscle cell proliferation in arteries due to the
development of an autocrine neointimal system for CNP that modulates
neointimal growth. The present study demonstrates the production and
secretion of CNP in rat vascular smooth muscle cells in the absence of
endothelium. In addition, these cells express atrial natriuretic
peptide (ANP) and the natriuretic peptide receptors A, B, and C. The
production and secretion of CNP in vascular smooth muscle cells is
stimulated by transforming growth factor-
, whereas basic fibroblast
growth factor plays an inhibitory role. These data show that ANP and mainly CNP are coexpressed with the natriuretic peptide receptors in
rat vascular smooth muscle cells. This provides evidence for a vascular
natriuretic peptide autocrine system of physiological relevance in
these cells.
atrial natriuretic peptide; brain natriuretic peptide; transforming
growth factor-; vascular smooth muscle
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INTRODUCTION |
NATRIURETIC PEPTIDES
have been shown to modulate several physiological functions. Atrial
natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have been
shown to elicit renal and cardiovascular effects (1, 23).
The third type, C-type natriuretic peptide (CNP), elicits mainly
vasodilatory effects rather than regulation of body fluid homeostasis
(33). The different physiological effects of these
peptides could be attributed to the existence of three different
receptor subtypes, natriuretic peptide receptor-A, -B, and -C [NPR-A,
NPR-B, and NPR-C, respectively (25)]. NPR-A is a
guanylate cyclase-coupled receptor activated by ANP and BNP (25). NPR-B is also a guanylate cyclase-coupled receptor,
which specifically binds CNP (18). Finally, the NPR-C is
devoid of kinase and guanylate cyclase activity and activates G
proteins (24). NPR-C binds with high affinity to all three
natriuretic peptides, and its ability to recycle rapidly suggests that
it might also act as a clearance receptor to internalize and degrade circulating natriuretic peptides (21, 25).
Proliferation of vascular smooth muscle cells is an important response
of arteries to several vascular injuries, such as atherosclerosis or
restenosis after angioplasties. Cytokines and growth factors, released
by the injured vascular wall and activated platelets, stimulate
proliferation of the vascular smooth muscle cells (6); however, little is known about factors that limit proliferation of
these cells once initiated. Natriuretic peptides have been reported to
inhibit cell proliferation in several cell types, such as vascular
smooth muscle cells through the generation of cGMP (11).
In support of this hypothesis, nitric oxide, a cGMP-elevating agent,
elicits antiproliferative effects in vascular smooth muscle cells
(11). In vascular smooth muscle cells, CNP, which is
commonly referred as an endothelium-derived relaxing factor (3,
4, 11), has been presented as the most potent inhibitor of
growth and proliferation, an effect mediated by the occupation of the guanylate cyclase-coupled NPR-B (27). Consistent with
this, CNP has been shown to inhibit arterial intimal thickening in
vivo, most likely through inhibiting vascular smooth muscle
proliferation induced by vascular injury (2, 3, 10).
Here we show for the first time that CNP and also ANP and their
receptors are expressed in vascular smooth muscle cells in the absence
of endothelium and independently of endothelial regulation. In
addition, we investigated the regulation of CNP expression by cytokines
and growth factors.
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MATERIALS AND METHODS |
Materials.
Rats were from Charles River (Margate, UK). Stroke-prone spontaneously
hypertensive rats (SPSHR) were from the Animal Facility of the
University of Birmingham. Collagenase, dextran, fluorescein-conjugated anti-rabbit IgG antibody, rhodamine-conjugated anti-rabbit IgG antibody, rabbit monoclonal anti-
-smooth muscle actin antibody, and
elastase were from Sigma (Poole, Dorset, UK). Rabbit anti-von Willebrand factor (vWF) polyclonal antibody was from Dako (High Wycombe, UK). Penicillin, fetal calf serum, medium M199, SuperScript II
preamplification kit, and Hanks' balanced salt solution (HBSS) were
from GIBCO-BRL (Paisley, UK). Sep-Pak C18 cartridges were from Waters (Watford, UK). CNP radioimmunoassay and rabbit polyclonal CNP antibody were from Peninsula Laboratories (Merseyside, UK). Hybond-N, [32P]CTP, and rapid-hyb buffer were from
Amersham. Stratagene random-primer labeling kit was from Stratagene
(Cambridge, UK). Geneclean II kit was from Bio101 (Vista, CA).
MiniMessage Maker kit, transforming growth factor- (TGF-), tumor
necrosis factor- (TNF-), interleukin-1 (IL-1),
interferon-
(INF-), and basic fibroblast growth factor (bFGF)
were from R & D Systems (Abingdon, UK). Plasmid Midi kit was from
Qiagen (Dorking, UK). Fuji RX film was from Stuart Basset (Nottingham,
UK). All other reagents were of analytic grade.
Cell culture.
Aortic smooth muscle cell (ASMC) preparation was performed as described
previously (12). Briefly, young male Wistar rats (180-200 g) were killed by carbon dioxide asphyxiation and the aortas were removed. The aortas were incubated in 3 mg/ml collagenase in medium M199 supplemented with 100 U/ml penicillin for 30 min at
37°C in a shaker bath, and the tunica media was dissected from the
adventitia and endothelium. The suspension was then incubated in 1 mg/ml elastase in M199 for 10 min at 37°C and for a further 2 h
in 0.5 mg/ml elastase and 1.5 mg/ml collagenase in M199 at 37°C. The
suspension was centrifuged at 900 g for 4 min, and cells were then resuspended into 10 ml M199 + 10% fetal calf serum. The
cells were plated at 4 × 105 cells/ml and incubated
at 37°C in 5% carbon dioxide.
Cerebral vascular smooth muscle (CVSM) cells were isolated as
previously described (7). Briefly, a total of 10 preparations was obtained from groups of 12 or 24 male Wistar rats
weighing 120 g. Rat brains devoid of cerebellum were removed and
placed in cold 4°C HBSS without Ca2+ and
Mg2+, pH 7.4. The pial membranes were removed, and the
cerebral cortices cleaned of white matter were homogenized. The
homogenate was centrifuged, and the pellet was resuspended in HBSS
containing 15% dextran and 5% fetal bovine serum and centrifuged
again at 3,000 g. The microvessel pellet was resuspended in
HBSS and filtered through a 150- µm nylon mesh sieve. The purity of
the preparation was assessed by both phase-contrast and electron
microscopy along with immunostaining with FITC-IgG to -smooth muscle actin.
All studies were performed in cultured vascular smooth muscle cells at
passage 3, because at further passages a certain degree of
dedifferentiation was observed (data not shown) as described previously
(15).
RT-PCR.
The procedures were as we described previously (40).
Briefly, poly(A)+ RNA was obtained from vascular smooth
muscle cells, rat embryos, and heart using a MiniMessage Maker kit.
RNA was reverse transcribed with the SuperScript II preamplification
kit and subjected to PCR with rat gene-specific intron-spanning primers
described in Table 1. Target sequences
were amplified at the profile 94°C 4 min, 94°C 30 s, 58°C
30 s, 72°C 1 min, and 72°C 7 min by using the same amount of
cDNA for all primer sets. The RT-PCR products were all of expected
size. Negative controls were performed by omitting the RT step or cDNA
template from PCR amplification. To confirm the identity of
size-fractionated PCR products further, Southern blots were performed
as described previously (28). Briefly, gels were
denatured, neutralized, and transferred onto Hybond-N. Blots were fixed
by ultraviolet transillumination for 4 min and hybridized to a rat cDNA
fragment (internal to amplified region) being labeled with
[32P]CTP using a Stratagene random-primer labeling kit.
Hybridization was performed in rapid-hyb buffer for 2 h at 65°C.
Blots were washed twice in 2× standard saline citrate (SSC)-0.1%
sodium dodecyl sulfate (SDS) for 15 min at 20°C and then in 0.1 × SSC-0.1% SDS for 15 min at 65°C. Blots were then exposed to Fuji
type RX film for 20 min at 
80°C. Finally, RT-PCR products were
purified from agarose gels after electrophoresis using a Geneclean II
kit and subcloned into the pSPORT vector. The plasmid containing the
cDNA insert was prepared using a Plasmid Midi kit and sequenced on an
ABI 3T3 automated sequencer.
For semiquantitative RT-PCR, the cycle number was adjusted between 20 and 35 cycles to yield visible products within the range of linear
amplification and also a calibration curve was done by varying
quantities of cDNA template for a fixed cycle number (35 cycles).
Resulting RT-PCR products were run on agarose gel and visualized by
staining with ethidium bromide. Their densities were quantified by
using a phosphorimager (Molecular Dynamics, Sunnyvale, CA) and
normalized to that of RT-PCR product of the housekeeping GAPDH gene in
the same sample.
Immunocytochemistry.
Cells were seeded onto poly-L-lysine-coated glass
coverslips in 24-well plates and grown in M199 with 10% fetal calf
serum. Monolayers were washed three times in PBS, fixed for 5 min in 70% ethanol and 1% glacial acetic acid at room temperature, washed twice with PBS for 3 min each, and blocked with PBS containing 3% BSA.
Cells were then incubated for 2 h with either rabbit polyclonal CNP antibody, rabbit anti--smooth muscle actin monoclonal, or rabbit
anti-vWF polyclonal antibody diluted 1:100, 1:100, and 1:200,
respectively, in PBS containing 3% BSA. After three washes in PBS for
3 min each, cells were incubated with fluorescein-conjugated or
rhodamine-conjugated anti-rabbit IgG secondary antibody diluted 1:200
for 2 h at room temperature. The cells were then washed three
times for 3 min each as before. Slides were mounted with 50%
glycerol-50% PBS and visualized with an immunoflourescence microscope
(Olympus, London, UK).
Radioimmunoassay.
Radioimmunoassays were done on extracts of cultured ASMC and
conditioned media using a commercial CNP radioimmunoassay. Briefly, treated cells were harvested and studied under paired conditions with
cells treated by vehicle alone. After being cooled on ice, glacial
acetic acid was added (final concentration 1 M), and the cells were
homogenized. The homogenates were centrifuged at 10,000 g
for 10 min, and the supernatants were extracted using Sep-Pak C18 cartridges previously activated by addition of 4 ml of
acetonitrile followed by 4 ml of 0.1% (vol/vol) trifluoroacetic acid
(TFA). The cartridges were loaded with sample and then washed with 4 ml
of 0.1% TFA. CNP was eluted with 3 ml of acetonitrile-water-TFA (60:40:0.1 vol/vol/vol). The eluates were lyophilized and dissolved in
the assay buffer of a commercial radioimmunoassay for CNP.
Statistical analysis.
Analysis of statistical significance was performed using Student's
t-test. The significance level was P < 0.05.
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RESULTS |
Examination of the primary culture of Wistar rat ASMC.
Freshly dispersed ASMC from healthy adult Wistar rats were prepared and
plated out in cell culture as described previously (12).
To verify that the primary culture of ASMC was free of contamination
with endothelial cells, we analyzed several samples with an antibody
that specifically recognizes -smooth muscle actin, which is a
differentiation marker of smooth muscle cells and thus is suitable to
distinguish smooth muscle cells from other cells (31). As
shown in Fig. 1A,
immunofluorescence studies of primary culture of ASMC incubated with
-smooth muscle actin antibody revealed that all the cells in the
culture expressed -smooth muscle actin (n = 8). To
further investigate the absence of endothelial contamination in the
primary culture of ASMC, we investigated the expression of
VEGF-R2/Flk-1 receptor, which is expressed in endothelial cells
(40), in the primary culture of ASMC. As a positive
control for the expression of VEGF-R2/Flk-1, rat embryos were used as
an endothelium-rich tissue. As expected, expression of VEGF-R2/Flk-1
was clearly found in homogenates of rat embryos (Fig. 1B;
n = 6); however, it was undetectable in the primary
culture of ASMC (Fig. 1B; n = 6). The
negative control was performed by omitting the cDNA template from PCR
amplification (Fig. 1B). These results indicate that the
primary culture of ASMC used in this study was free of contamination
with endothelial cells.
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Fig. 1.
The primary culture of aortic smooth muscle cells
(ASMC) is free of contamination with endothelial cells. Ai:
ASMC were immunostained with both the rabbit anti--smooth muscle
actin monoclonal antibody and rabbit anti-von Willebrand factor
polyclonal antibody as described in MATERIALS AND METHODS
(n = 8). Aii: phase-contrast micrograph of
the immunofluorescent image of ASMC. Scale bar represents 10 µm.
B: comparison of VEGF-R2/Flk-1 transcripts in rat
embryo and culture ASMC. VEGF-R2/Flk-1 (408 bp) specific PCR products
as well as GAPDH specific transcripts (400 bp) were separated by
agarose gel electrophoresis and further processed by Southern blot
hybridization as described in MATERIALS AND METHODS.
For the amplification of VEGF-R2/Flk-1 and GAPDH sequences, 10 ng cDNA
was used. Negative controls were performed by omitting the cDNA
template from PCR amplification. The autoradiograms are representative
of 6 separate determinations.
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Detection of mRNAs coding for natriuretic peptides
and their receptors in primary culture of ASMC.
Representative autoradiograms show the presence of RNA transcripts for
ANP and CNP and for all three natriuretic peptide receptors in rat ASMC
(Fig. 2; n = 6). Rat ASMC
mRNA was extracted, and RT-PCR with Southern blot hybridization and
internal probing of gene fragments for the natriuretic peptides and
their receptors was performed as described in MATERIALS AND
METHODS. Heart tissue from Wistar rats served as control, because
ANP, BNP, and the natriuretic peptide receptors NPR-A, -B, and -C are
expressed in this tissue (see Refs. 22, 26).
As shown in Fig. 2, ANP mRNA was found in both ASMC and heart. ANP mRNA
expression, shown as a percentage of that for GAPDH, was 27 ± 5 in ASMC vs. 56 ± 10 in heart (P < 0.01;
n = 6). In contrast, BNP could only be detected in
Wistar heart (21.7 ± 6% of GAPDH expression) and was not found
at either passage 3 (Fig. 2; n = 6) or
successive passages (data not shown) of cultured ASMC. As reported
previously, CNP was not expressed in heart tissue from Wistar rat
(20), but we have detected CNP mRNA in ASMC (Fig. 2;
32 ± 8% of GAPDH expression; n = 6). As reported
previously, NPR-A, NPR-B, and NPR-C were found to be expressed in the
heart of Wistar rats (26), and, as shown in Fig. 2, these
receptors are also expressed in ASMC. mRNA expression for NPR-A, NPR-B,
and NPR-C, shown as a percentage of that for GAPDH, were 17 ± 4, 98 ± 12, and 57 ± 11, respectively, in ASMC. In
heart tissue we found similar results for NPR-A mRNA expression (12 ± 4% of GAPDH expression), greater levels of NPR-B mRNA
(152 ± 21; P < 0.01), and lower levels of NPR-C
mRNA (21 ± 7; P < 0.01; n = 6).
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Fig. 2.
Representative RT-PCR of cDNA sequences of natriuretic
peptides and their receptors in cultured rat ASMC and rat heart. ASMC
or heart poly(A)+ RNA (0.1 µg) was reverse transcribed
and subjected to 35 cycles of PCR using 2 specific primers.
Corresponding amplification products were size fractionated on 1.2%
agarose gel electrophoresis and analyzed by Southern blot hybridization
as described in MATERIALS AND METHODS. GAPDH was used as an
internal positive control. Each autoradiogram is representative of 6 separate determinations.
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Effect of various agents on CNP mRNA
expression in rat ASMC.
We examined the effects of different agents that affect vascular tone
and growth, such as the cytokines IFN-, TNF-, and IL-1, or the
growth factors TGF- and bFGF, on the regulation of CNP expression in
ASMC. Primary culture of Wistar rat ASMC at passage 3 was
treated with IFN- (100 ng/ml), TNF- (100 ng/ml), IL-1 (100 ng/ml), bFGF (100 ng/ml), or TGF- (500 pM), and CNP mRNA expression
was evaluated with RT-PCR. GAPDH was PCR amplified simultaneously
with CNP and used as an internal control.
Treatment of cultured ASMC for 24 h with the cytokines IFN-,
TNF-, or IL-1 did not significantly alter CNP mRNA expression (Table 2 and Fig.
3; n = 6). In
contrast, treatment for 24 h with the growth factors TGF- or
bFGF significantly modified CNP mRNA expression in cultured ASMC. As
shown in Table 2 and Fig. 3, incubation with TGF- (500 pM) for
24 h significantly increases CNP mRNA expression compared with
control (nontreated cells; n = 6; P < 0.01); however, bFGF had opposite effects. Treatment with 100 ng/ml
bFGF for 24 h resulted in a significant reduction in CNP mRNA
expression in ASMC compared with vehicle-treated cells (Table 2 and
Fig. 3; n = 6; P < 0.01).
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Fig. 3.
C-type natriuretic peptide (CNP) mRNA expression in ASMC
(A) or cerebral smooth muscle cells (CVSM; B)
following treatment with different agents. ASMC or CVSM cells were
treated for 24 h in the presence of basic fibroblast growth factor
(bFGF; 100 ng/ml), transforming growth factor (TGF)- (500 pM),
interferon (IFN)- (100 ng/ml), tumor necrosis factor (TNF)- (100 ng/ml), interleukin (IL)-1 (100 ng/ml), or the vehicles (Control) as
indicated. mRNA preparation, RT-PCR with GAPDH internal positive
control, Southern blot hybridization, and radiolabeled probing were
performed as described in MATERIALS AND METHODS. Each
autoradiogram is representative of 6 separate
determinations.
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The effect of TGF- on CNP expression in cultured Wistar rat ASMC was
concentration dependent. Treatment of ASMC for 24 h with TGF-
increases CNP expression, reaching a maximal effect at 500 pM with
4.3 ± 0.4-fold increase. We found that incubation of ASMC with
higher concentrations of TGF- induced smaller increases in CNP
expression (Fig. 4).
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Fig. 4.
Concentration-dependence relationship of CNP
expression in response to TGF- in cultured rat ASMC. A:
rat ASMC were treated for 24 h in the presence of various
concentrations of TGF- (100-2,000 pM) or the vehicle as
indicated. RT-PCR with GAPDH internal positive control, Southern blot
hybridization, and radiolabeled probing were performed as described in
MATERIALS AND METHODS. The autoradiograms are
representative of 8 separate determinations. B: histograms
indicating CNP mRNA expression after the treatment with different
concentrations of TGF- relative to that for GAPDH and represented as
percentage of control (vehicle was added). Values are means ± SE
of 8 representative experiments.
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The effect of TGF- on CNP expression in ASMC was confirmed by
immunocytochemistry. Cultured Wistar rat ASMC were treated with TGF-
(500 or 2,000 pM) for 24 h, and CNP was detected using an anti-CNP
polyclonal antibody. In agreement with the results reported above,
fluorescent microscopy of ASMC revealed that treatment with TGF- for
24 h induced a biphasic increase in fluorescence compared with
untreated cells, indicating that TGF- stimulated CNP expression in
ASMC (Fig. 5; n = 8). The
negative controls (cells incubated with anti-CNP polyclonal
antibody saturated with CNP 1 µM) reveal that fluorescence
intensity was indicative of CNP expression (Fig. 5).
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Fig. 5.
Immunofluorescence micrographs of fixed cultured rat ASMC
stained with rabbit anti-CNP antibody. ASMC were treated for 24 h
in the absence (A) or presence of 500 pM TGF-
(B) or 2,000 pM TGF- (C) and then fixed. Cells
were then immunostained with the rabbit polyclonal anti-CNP antibody or
with anti-CNP polyclonal antibody saturated with CNP 1 µM
(D) followed by incubation with fluorescein-conjugated
anti-rabbit IgG antibody as described in MATERIALS AND
METHODS (n = 8). E-H: phase-contrast
micrograph of the immunofluorescent images of ASMC. Scale bar
represents 20 µm.
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Effect of various agents on CNP mRNA
expression in rat CVSM cells.
To further investigate the effect of TGF-, bFGF, IFN-, TNF-,
and IL-1 on CNP expression in vascular smooth muscle cells, we
examined their effects in cultured CVSM cells from Wistar rats.
CVSM cells at passage 3 in culture were treated for 24 h with bFGF (100 ng/ml), TGF- (500 pM), IFN- (100 ng/ml), TNF-
(100 ng/ml), or IL-1 (100 ng/ml), and CNP mRNA expression was
evaluated as described in MATERIALS AND METHODS. GAPDH was
PCR amplified simultaneously with CNP and used as an internal control.
Consistent with the results obtained in ASMC, treatment of CVSM cells
for 24 h with TGF- (500 pM) significantly increases CNP mRNA
expression (Table 3 and Fig. 3;
n = 6; P < 0.01). In addition,
incubation with bFGF (100 ng/ml) significantly decreases CNP mRNA
expression (Table 3 and Fig. 3; n = 6;
P < 0.01). No significant modifications were detected
when CVSM were treated with IFN-, TNF-, or IL-1 as reported
for ASMC (Table 3 and Fig. 3; n = 6).
In agreement with the results reported above, the effect of TGF- on
CNP expression was confirmed by immunocytochemistry. Treatment of CVSM
cells with TGF- (500 or 2,000 pM) for 24 h induced a biphasic
increase in CNP immunodetected compared with untreated cells,
indicating that TGF- stimulated CNP expression in CVSM cells (Fig.
6; n = 8). The negative
controls (CVSM cells immunostained with anti-CNP polyclonal antibody
saturated with CNP 1 µM) reveal that fluorescence was indicative of
CNP expression (Fig. 6). These findings reveal for the first time the
expression of CNP in smooth muscle cells from rat aorta or cerebral
vasculature.
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Fig. 6.
Immunofluorescence micrographs of fixed cultured rat CVSM
stained with rabbit anti-CNP antibody. CVSM were treated for 24 h
in the absence (A) or presence of 500 pM TGF-
(B) or 2,000 pM TGF- (C) and then fixed. Cells
were then immunostained with the rabbit polyclonal anti-CNP antibody or
with anti-CNP polyclonal antibody saturated with CNP 1 µM
(D) followed by incubation with fluorescein-conjugated
anti-rabbit IgG antibody as described in MATERIALS AND
METHODS (n = 8). E-H: phase-contrast
micrograph of the immunofluorescent images of CVSM. Scale bar
represents 10 µm.
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Regulation of CNP secretion by TGF- and bFGF in Wistar and SPSHR
rat ASMC and CVSM.
The effect of TGF- on CNP mRNA expression was confirmed by
radioimmunoassay analysis of CNP in ASMC and CVSM cells in culture. As
reported above, TGF- induced a biphasic increase in CNP expression in ASMC. Stimulation with maximal concentration of TGF- (500 pM)
induced a significant increase in the level of CNP expressed, whereas
incubation with a supramaximal concentration (2,000 pM) did not induce
any detectable increase over the basal level (Table 4;
n = 8). Similar results were observed in CVSM (Table
4; n = 8). Consistent
with the effect of bFGF on CNP mRNA expression reported above,
treatment of ASMC or CVSM with 100 ng/ml bFGF induced a slight decrease
in CNP-like immunoreactivity and significantly reduced CNP expression
stimulated by 500 pM TGF- (Table 4; n = 8).
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Table 4.
Changes in CNP-like immunoreactivity in Wistar and SPSHR aortic and
cerebral smooth muscle cells and medium in response to TGF- and bFGF
treatment as measured by radioimmunoassay
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TGF- has been shown to stimulate CNP secretion in endothelial cells
in a concentration-dependent manner (36). Hence the effect
of TGF- on CNP secretion in ASMC and CVSM was investigated. Table 4
shows the effect of incubation for 24 h with 500 and 2,000 pM
TGF- on CNP concentration in ASMC- and CVSM-conditioned media. As
reported in endothelial cells (36), TGF- stimulated accumulation of CNP-like immunoreactivity in ASMC- and CVSM-conditioned media in a concentration-dependent manner (Table 4; n = 8). In contrast, treatment with bFGF (100 ng/ml) did not induce any
detectable increase in CNP-like immunoreactivity (Table 4;
n = 8) and simultaneous stimulation with bFGF (100 ng/ml) and TGF- (500 pM) induced an increase in CNP-like
immunoreactivity significantly smaller than that achieved by TGF-
alone (Table 4; n = 8).
The effect of TGF- and bFGF on CNP production and secretion was also
examined in ASMC and CVSM cells from SPSHR, which present a higher
level of TGF- mRNA than in normotensive smooth muscle cells
(14). ASMC from SPSHR were obtained and cultured following the same protocol used for ASMC from Wistar rats. Treatment for 24 h with the growth factors TGF- or bFGF significantly modifies CNP
mRNA expression in cultured ASMC or CVSM from SPSHR. As shown in Table
5, incubation with TGF- (500 or 2,000 pM) for 24 h significantly increases CNP mRNA expression compared
with control in a linear concentration-dependent manner
(n = 6; P < 0.01). Different from in
normotensive Wistar rats, treatment with 100 ng/ml bFGF for 24 h
resulted in a significant increase in CNP mRNA expression in both ASMC
and CVSM from SPSHR compared with vehicle-treated cells (Table 5;
n = 6; P < 0.01). As shown in Table 4,
in ASMC and CVSM cells from SPSHR, TGF- induced a
concentration-dependent increase in CNP production. Consistent with the
results obtained when CNP mRNA was examined, we found that TGF-
increases CNP protein level in the cells without the biphasic effect
observed in ASMC from normotensive rats at the same range of
concentrations. bFGF induced a significant increase in CNP protein
level in ASMC and CVSM from SPSHR and potentiated the effect of TGF-
(500 pM) when incubated simultaneously (Table 4; n = 8). ASMC and CVSM cells from SPSHR showed a higher basal level of
CNP-like immunoreactivity in the media. As in normotensive Wistar rat
smooth muscle cells, treatment for 24 h with TGF- induced a
significant increase in CNP-like immunoreactivity in ASMC- and
CVSM-conditioned media (Table 4; n = 8). In contrast,
treatment with bFGF (100 ng/ml) did not induce either increased CNP
secretion (Table 4; n = 8) or reduction of
TGF--stimulated increase in CNP-like immunoreactivity (Table 4;
n = 8).
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Table 5.
CNP mRNA expression in aortic and cerebral smooth muscle cells from
SPSHR following treatment with bFGF and TGF-
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DISCUSSION |
The present study demonstrates the expression and secretion of CNP
in vascular smooth muscle cells and their regulation by the growth
factors TGF- and bFGF. CNP, a peptide of 22 or 53 amino acid
residues initially isolated from the porcine brain (34),
has also been shown to be expressed and secreted by endothelial cells,
suggesting a significant role in the modulation of vascular tone and
proliferation in an antagonistic manner to the renin-angiotensin system
(36). Our data show the presence of specific mRNAs coding for the ANP and predominantly CNP in aortic vascular smooth muscle cells from rat. In addition, the expression of NPR-A, -B, and -C was
simultaneously detected in rat ASMC. The presence of NPR-B and CNP,
believed to be the specific ligand for NPR-B (18), suggests a role for CNP as an autocrine and/or paracrine regulator of
smooth muscle cell growth and vascular tone independently or in concert
with endothelial cells.
The production of CNP in vascular smooth muscle cells was corroborated
in vascular smooth muscle cells from cerebral microvessels. In these
cells, as well as in ASMC, we found that CNP mRNA expression is
modulated by growth factors such as TGF- and bFGF.
Endothelium-secreted CNP has been reported to inhibit vascular smooth
muscle cell proliferation (19), but after arterial injury,
in regions stripped of endothelium, smooth muscle cell proliferation is
only transiently stimulated, resulting in development of neointima,
which consists mainly of modified vascular smooth muscle cells that
become a new source of CNP in the absence of endothelium
(3). Our results reporting the expression of CNP in
cultured vascular smooth muscle cells support the hypothesis that these
cells develop an autocrine system for CNP that regulates neointimal
proliferation independently or in concert with the endothelial cells.
Proliferation and migration of vascular smooth muscle cells with
subsequent intimal thickening is a major process in the development of
restenosis after angioplasty or atherosclerosis. Several growth factors
have been shown to be involved in these events. In agreement with this,
TGF- is a growth inhibitor of endothelial cells (32) and a potent stimulator of CNP secretion (36). In vascular
smooth muscle cells TGF- is a growth modulator (32),
and, consistent with the effect on endothelial cells, our results
indicate that TGF- stimulates CNP expression and secretion in these
cells. We found that the effect of TGF- on CNP mRNA expression is
concentration dependent, reaching a maximum at 500 pM. At higher
concentrations, TGF- induced smaller increases in CNP mRNA and
protein level. These results suggest that supramaximal doses of TGF-
are less effective at stimulating CNP production, which highlights the significance of the concentration of TGF- in the vascular tissues in
the regulation of smooth muscle cell proliferation. On the other hand,
TGF- stimulates CNP secretion in a linear concentration-dependent manner. The fact that high doses of TGF- stimulate CNP secretion while having little effect in production might explain why CNP protein
levels in cells treated with TGF- (2,000 pM) are smaller than in
nonstimulated cells. In agreement with the effect reported in
endothelial cells (36), the stimulatory effect of TGF-
on CNP production and secretion raises the possibility that the role of
TGF- on vascular smooth muscle growth might be mediated by increases
in CNP secretion.
Other growth factors, such as bFGF, have been shown to stimulate CNP
secretion in endothelial cells (35, 36). In contrast, our
present observations clearly show that incubation with bFGF inhibited
the expression of CNP mRNA in both aortic and CVSM cells. In addition,
treatment of ASMC with bFGF reduced TGF--stimulated production and
secretion of CNP.
Abnormal vascular smooth muscle proliferation is considered to be one
of the factors contributing to increased peripheral vascular resistance
in hypertension. Proliferation of cultured ASMC from SPSHR has been
reported to be increased in response to treatment with several growth
factors (13, 30, 41). The accumulation of TGF- mRNA
levels is higher in SPSHR smooth muscle cells than in normotensive
Wistar-Kyoto (WKY) rats, and DNA synthesis is enhanced in response to
exogenous TGF-1 in SPSHR, although it does not have any
detectable effect in WKY rats (14). In the present study
we show that the actions of TGF- and bFGF on CNP production and
secretion are altered in SPSHR compared with normotensive rats. Our
results indicate that CNP mRNA expression and protein level in the
cells are not reduced when supramaximal concentrations of TGF- were
used to stimulate ASMC or CVSM from SPSHR, whereas in normotensive
rats, those concentrations had little effect in CNP production.
Although we have not further investigated this event, the data
presented suggest that the relationship between TGF- and CNP
production in vascular smooth muscle cells from SPSHR could be linear
instead of the biphasic pattern observed in normotensive rats. In
contrast to the inhibitory role of bFGF on CNP expression in
normotensive rats, we found that bFGF stimulated CNP production in ASMC
and CVSM cells from SPSHC. Consistent with this, the combined effect of
TGF- and bFGF was found to be additive. In addition, our data
clearly show an elevated basal CNP secretion in ASMC and CVSM cells
from SPSHR compared with normotensive rats, which might be a response
to compensate for the hypertension. Although the cause of these
differences is not well understood, the regulation of CNP production
and secretion in ASMC and CVSM cells from SPSHR is altered and we do
not exclude the possibility that the role of CNP in the regulation of
vascular smooth muscle cell proliferation in these rats might be
different from that in normotensive rats.
Cytokines have been reported to stimulate CNP production and secretion
in endothelial cells (35). In contrast, we found that
stimulation of ASMC or CVSM with either IFN-, TNF-, or IL-1
did not significantly modify CNP mRNA expression in these cells,
indicating that CNP secretion is not modulated by cytokines in rat
vascular smooth muscle cells.
In conclusion, we have shown the presence of transcripts of ANP and,
predominantly, CNP as well as mRNAs coding for all three natriuretic
peptide receptors in cultured rat vascular smooth muscle cells. These
data provide evidence for the existence of a vascular natriuretic
peptide system that may serve as a local control to modulate vascular
growth and tone, either alone or in concert with the endothelial cells.
Perspectives
It has been proposed that natriuretic peptides can act not only as
vasodilators but also as growth inhibitors of vascular smooth muscle
cells (36). In the present study we reported the expression of the natriuretic peptides ANP and CNP as well as the
receptors NPR-A, -B, and -C in vascular smooth muscle cells. We found
that production and secretion of CNP, which induces inhibition of
vascular smooth muscle cell proliferation (39), is
augmented by TGF-, whereas opposite effects were exerted by bFGF. As
well as in endothelial cells, TGF- has been presented as a growth regulator of vascular smooth muscle cells (32). In
endothelial cells, this effect has been suggested to be mediated by
stimulation of CNP secretion (36). The findings presented
here raise the possibility that the role of TGF- in vascular smooth
muscle growth might be mediated by the modulation of CNP production and
secretion. Studies performed in SPSHR vascular smooth muscle
cells have demonstrated that the regulation of CNP production and
secretion by growth factors are altered and the basal levels of CNP
production and secretion are higher than in normotensive rats. Although
speculative, the different regulation of CNP in SPSHR might be a
response to compensate for the factors contributing to increased
peripheral vascular resistance in hypertension. Taken together these
results support the existence of a vascular natriuretic peptide system where endothelial and vascular CNP production might induce vascular relaxation and growth inhibition in a paracrine or autocrine manner.
| |
ACKNOWLEDGEMENTS |
This work was supported by The British Heart Foundation.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: G. E. Woodard, Physiological Laboratory, Downing St., Univ. of Cambridge, Cambridge CB2 3EG, UK (E-mail:
GeoffreyW{at}intra.niddk.nih.gov).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 May 2001; accepted in final form 10 September 2001.
| |
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